Figure 6.

CipA lacks intrinsic proteolytic activity on C3b and C4b and formation of a quadripartite complex to inactivate C3b and C4b. Intrinsic proteolytic activity of CipA in FI-mediated inactivation of C3b was assessed by Western blotting (A–C). CipA or BSA coated wells were incubated with C3b or with C3b and FI for 1 h (A) and 6 h (B) at RT or for 6 h at 37°C (C). After termination, C3b degradation products were visualized by Western blotting employing a polyclonal anti-C3 antibody. Detection of CipA (D) in reaction mixtures shown in (C) by using an anti-CipA antibody (23). Formation of a quadripartite complex results in inactivation of C3b (E) and C4b (F). Microtiter plates coated with CipA, CipA ΔE360-K369 or CipA E360P (5 ng/µl) were incubated in the absence (-FI) or presence of FI (+FI). Thereafter, reaction mixtures containing C3b and FH or C4b and C4BP were added to the wells and were then subjected to SDS-PAGE and Western blotting. C3b and C4b degradation products were visualized by using an anti-C3 antibody and a mixture containing an anti-C4 and anti-C4d antibody. Control reactions containing C3b, FI, and FH as well as C4b, FI, and C4BP.